THE PROXIMAL LIGAND VARIANT HIS93TYR OF HORSE HEART MYOGLOBIN
THE PROXIMAL LIGAND VARIANT HIS93TYR OF HORSE HEART MYOGLOBIN
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DOI：
10.1021/bi00006a021
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发表时间：
1995-02-14
期刊：
影响因子：
2.9
通讯作者：
MAUK, AG
中科院分区：
文献类型：
--
作者：
HILDEBRAND, DP;BURK, DL;MAUK, AG
人们对马心肌红蛋白 His93Tyr 变体的光谱和结构特性进行了研究，以评估用酪氨酰残基取代该蛋白质的近端 His 残基（如不同来源的过氧化氢酶中发生的情况）的效果。三价铁形式的变体表现出与 pH 值 7 至 10 之间的 pH 值无关的电子光谱，并且其吸收最大值和强度的变化与活性位点的五配位血红素铁中心一致。该变体的 EPR 谱是高自旋、菱形系统的谱，与报道的牛肝过氧化氢酶的谱相似。该变体的 1D H-1-NMR 谱证实了血红素铁中心的五配位性质，并在 112.5 ppm 处表现出广泛的共振，这归因于酚盐配体的间位质子。该结果表明新的 Tyr 配体在该蛋白质中以显着的速率翻转。 Fe(III) 衍生物的热稳定性与野生型蛋白 (pH 8) 相比没有变化，而中点还原电位 [-208 mV vs SHE（pH 8.0, 25 摄氏度）] 低约 250 mV。通过 X 射线衍射分析确定的变体的三维结构证实了血红素铁中心的五配位性质，并确定近端 Tyr 配体的引入是通过 F 螺旋（残基 88-99）的移动来实现的，其中该残基远离血红素袋。这种变化的额外影响是 Leu29、血红素丙酸酯和血红素乙烯基的位置发生小幅变化，并伴随着与血红素辅基的氢键相互作用的改变。 Tyr93 残基相对于血红素基团的位置也不同于通常存在的 His93 残基的位置，并且类似于牛肝过氧化氢酶的近端 Tyr 残基的位置。
The spectroscopic and structural properties of the His93Tyr variant of horse heart myoglobin have been studied to assess the effects of replacing the proximal His residue of this protein with a tyrosyl residue as occurs in catalases from various sources. The variant in the ferric form exhibits electronic spectra that are independent of pH between pH 7 and 10, and it exhibits changes in absorption maxima and intensity that are consistent with a five-coordinate heme iron center at the active site. The EPR spectrum of the variant is that of a high-spin, rhombic system similar to that reported for bovine liver catalase. The 1D H-1-NMR spectrum of the variant confirms the five-coordinate nature of the heme iron center and exhibits a broad resonance at 112.5 ppm that is attributable to the meta protons of the phenolate ligand. This result indicates that the new Tyr ligand flips at a significant rate in this protein. The thermal stability of the Fe(III) derivative is unchanged from that of the wild-type protein (pH 8) while the midpoint reduction potential [-208 mV vs SHE (pH 8.0, 25 degrees C)] is about 250 mV lower. The three-dimensional structure of the variant determined by X-ray diffraction analysis confirms the five-coordinate nature of the heme iron center and establishes that the introduction of a proximal Tyr ligand is accommodated by a shift of the F helix (residues 88-99) in which this residue resides away from the heme pocket. Additional effects of this change are small shifts in the positions of Leu29, a heme propionate, and a heme vinyl group that are accompanied by altered hydrogen bonding interactions with the heme prosthetic group. The position of the Tyr93 residue with respect to the heme group is also different from that of the His93 residue normally present and resembles that of the proximal Tyr residue of bovine liver catalase.